High quality reagents for innate immunity research, vaccine development and drug discovery

		Frequently asked questions relating to the Phytotitre library Please scroll down the page to view answers to each of the following questions: 1) What are the advantages and disadvantages of plant extract libraries compared to synthetic molecule libraries? 2) Won’t I just discover known compounds? Will I be able to generate IP? 3) What is the rationale for the choice of species comprising the library? 4) Will I be able to keep intellectual property (IP) I develop through use of the library? 5) How many unique molecules are contained in each extract of the library? 6) What ‘hit-rate’ can I expect from the library? 7) What are the advantages and disadvantages of the Phytotire plant extract library relative to larger plant extract libraries? 8) How rapidly can you resupply herbs or extracts for follow-on studies? 9) Can you supply unextracted plant material? 10) Can you tell me exactly how the extracts are prepared so I can take hits forward myself? 11) Where can I find information on the ethnopharmacological use of the products of interest? 12) Will I need a robot to screen the Phytotitre library? 13) What is the shelf life of the library? 14) Can you offer help or advice on how to identify active compounds in ‘hit’ extracts? 15) Are the extracts provided in the library suitable for use in human or animal studies? 16) To what extent has the Phytotitre library been pre-fractionated? 17) Have you removed molecules which can be problematic for some screening approaches? 1) What are the advantages and disadvantages of plant extract libraries compared to synthetic molecule libraries? For a more expansive discussion of the factors affecting the decision to choose a plant extract or a synthetic compound library, please click here. Generally speaking however, synthetic compound libraries offer the advantage of a more rapid progression from hit discovery to the lead optimisation stage, since every compound in the library is of known structure and means of synthesis. By contrast, phytochemicals occupy a chemical space with a far greater structural diversity than synthetic compound libraries, and tend to be more ‘drug-like’, with superior ADME/T (absorption, distribution, metabolism, excretion and toxicity) properties. Plants have also faced millennia of intense selective pressure to develop secondary metabolites that target specific pathways in microbes or herbivores, including mammals. As a result, the typical ‘hit-rate’ of natural product library screens tends to much higher than those yielded by synthetic libraries. For this reason, and because plant extracts contain hundreds of compounds per well, the early screening stages of plant extract libraries are much less costly in terms of labour, time and reagents, than equivalent screens of synthetic compound libraries. 2) Won’t I just discover known compounds? Will I be able to generate IP? This topic is covered in more depth in the associated guide. Briefly, however, the Phytotitre library contains many plants which have been very little studied, and therefore has potential for the discovery of new compounds. Although many of the hits from this library may turn out to be established compounds, and are therefore not directly patentable, derivatives of these compounds or their scaffolds offer excellent opportunities for IP generation, especially if a means of synthesis can be described. This model has yielded some of the most profitable drugs of all time (e.g. the statins, with global sales of >$130 Bn), and continues to be a fruitful source of IP (>50% of new drugs from 1981-2010 were nature-inspired). [1] In terms of novelty, the greater emphasis should be placed on the new target and assay. To date, only about 400 pharmacological targets have been screened and successfully drugged. By contrast, tens of thousands of potential protein targets and their partner interactions have never been screened for modulation by small molecules before. These targets, and particularly the bioassays developed to explore or quantify their function, offer vast potential for new drug discovery. The novelty of any new screening programme therefore depends not so much the library, but rather the discovery of a new potential target that may be linked to disease or another relevant phenotype, and the establishment of a bioassay to measure the function of that target. In other words, the key value and novelty are to be found in your new target and assay. Phytotitre aims to bring accessible high-quality screening to these new targets and assays. [1] Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 75:311-335 (2012) 3) What is the rationale for the choice of species comprising the library? The library has been designed to maximise potential content of molecules that may interact with biological pathways of interest, while minimising content of potentially toxic compounds. Only plants with a history of oral or topical medicinal use in man, or a history of inverse association with risk of disease, are included (239 traditional medicines and 128 commonly consumed plants). While some of the plants are well studied, others are poorly characterised, offering useful potential for the discovery of novel compounds. The library has been sized to optimally balance accessibility and workflow for smaller research groups, together with sufficient representation of phytochemical diversity. A list of all plants included in the library, together with notes on the rationale for the inclusion of each, based on either ethnopharmacological use and/or evidence of association with disease in epidemiological or interventional studies, is provided with all versions of the library (please click here to view a sample page). 4) Will I be able to keep intellectual property (IP) I develop through use of the library? Yes, all intellectual property (IP) that you generate through use of the library will belong to you. No licensing or material transfer agreements (MTA) are required to use the library. 5) How many unique molecules are contained in each extract of the library? This question is much debated in the field, and a consensus answer is yet to emerge. However, estimates of the average number of distinct compounds present within a crude plant extract range from hundreds to thousands [1]. The entire Phytotitre library may therefore contain thousands to hundreds of thousands of structurally distinct compounds - far greater than a synthetic compound library of equivalent size. [1] Hostettmann K. Strategy for the Biological and Chemical Evaluation of Plant Extracts. IUPAC (1999) 6) What ‘hit-rate’ can I expect from the library? The hit rates of screening campaigns vary considerably depending on the target, the variability of the bioassay and the signal to background ratio of the reporter used. However, as a general rule, the screening of natural product libraries tends to offer a far higher hit rate than synthetic chemical libraries. For example, screens of ~7,000 natural product-derived polyketide metabolites have yielded 20 commericalised drugs (an overall hit rate of 0.3%, all the way through to approval and commercialisation) [1]. By contrast, the hit rate of HTS of synthetic libraries can be lower than 0.001%. [1] It is impossible to estimate how many hits you will generate using your own assay to screen the Phytotitre library. However, hit rates of 0.1% to 1% are not uncommon for cell- or protein-based screening assays of natural product extract libraries after counterscreen triage. [1] Weissman KJ, Leadlay PF. Combinatorial biosynthesis of reduced polyketides. Nat Rev Microbiol 3:925-36 (2005) 7) What are the advantages and disadvantages of the Phytotire plant extract library relative to larger plant extract libraries? Most natural product libraries have been developed with the aim of capturing the greatest possible geographical and biochemical diversity and, as a result, are very large (thousands of extracts and hundreds of plates). While this approach provides a good chance of identifying dozens of hits, the costs of screening such libraries, particularly in terms of labour, time and reagents, are far beyond the means of most small research groups. Fortunately, it is now recognised that many phytochemicals are expressed widely across species of the same genus, and it has been shown that a broad sampling of biodiversity may not be essential for successful natural product-based drug discovery [1]. Indeed, a recent analysis of all alkaloids in medical use today showed that 93% of these molecules occurred more than 50 times in the Global Biodiversity Information Facility (GBIF) database, and only two had less than 10 occurences [2]. These observations indicate that an acceptable hit-rate may be obtained from modestly sized natural product libraries. The Phytotitre library has been carefully sized to optimally balance high molecular diversity and potential for lead discovery with appropriate workflow by independent research groups. By focusing only on plants with a history of use as traditional medicines, or association with reduced risk of disease in human dietary and epidemiological studies, the Phytotitre library furthermore seeks to maximise the potential hit-rate per time and reagent input, while at the same time minimising risk of identifying molecules of excessive toxicity. Further discussion of this point is available here. [1] Tulp M, Bohlin L. Functional versus chemical diversity: is biodiversity important for drug discovery? Trends Pharmacol Sci 5:225-231 (2002) [2] Amirkia V, Heinrich M. Alkaloids as drug leads - a predictive structural and biodiversity-based analysis. Phytochem Lett 10:xlviii-liii (2014) 8) How rapidly can you resupply herbs or extracts for follow-on studies? We will be happy to supply dried, unextracted material from any of the plants included in the Phytotitre library. We can also supply gram quantities of extracts in the absence of DMSO to facillitate dereplication and structural identification of lead compounds. 9) Can you supply unextracted plant material? Because none of the plants in the library are rare or endangered, and all are commercially available, there are no issues with recollection, sustainability, Nagoya protocol conventions or sovereignty of genetic resources. We will be happy to point you towards suitable suppliers for raw materials to take your hits forward at larger scale if necessary. 10) Can you tell me exactly how the extracts are prepared so I can take hits forward myself? Yes, the methods for each type of extraction are provided here. 11) Where can I find information on the ethnopharmacological use of the products of interest? The plant extract database provided with each kit includes examples of ethnomedicinal uses of each product, together with a summary of reported effects on human health or animal models of disease (if applicable) for certain plants. A sample page can be viewed by clicking here. 12) Will I need a robot to screen the Phytotitre library? The library is designed to be robot compatible, but it can also be screened manually by a single operator with the use of a multi-channel pipette, or in lower throughput formats if desired. 13) What is the shelf life of the library? We recommend that the kit is used within 6 months of receipt if stored at -20oC and two years if stored at -80oC. Aliquoting to daughter plates is recommended if multiple freeze thaw cycles are anticipated. We recommend warming to room temperature for at least 2 hours after defrosting before removing plate seals to avoid water absorption by DMSO, since the accumulation of moisture will accelerate the degradation of compound activity. 14) Can you offer help or advice on how to identify active compounds in ‘hit’ extracts? If you are new to the separation and identification of active compounds from plant extracts, please click here to view our resources page which includes sample methods and links to online resources which may assist your progression from hit discovery to the structural identification of lead compounds. We will be also happy to put you in touch with our partner organisation to help you with activity guided separation and compound identification if required. 15) Are the extracts provided in the library suitable for use in human or animal studies? No, not directly. The extracts provided in the library are provided as solutions in DMSO. The library is provided for in vitro research purposes only, and the constituents are not intended for human or animal consumption. However, the plant species comprising the library have been chosen to maximise the opportunity for rapid translation to human or animal dietary or nutraceutical studies. In particular, the majority of the plants included have a history of safe oral use in man. We anticipate that this approach may enable preliminary information to be gained rapidly with respect to the bioavailability and pharmacokinetics of lead compounds through the use of the parent raw plant materials in feeding studies. 16) To what extent has the Phytotitre library been pre-fractionated? A balance needs to be struck between the number of fractions of each extract used to prepare a natural product library prior to screening, and the costs of the subsequent screening efforts. Pre-fractionation can reduce interference between compounds within samples, so increasing the hit-rate, and simplifies subsequent activity-guided separation. However, increasing the number of pre-fractions also increases the number of plates that must be screened, and accordingly the labour, time and cost of the screening component of the work. Phytotitre provides two pre-fractions per sample, one polar and one non-polar, since this balances optimum library size with ease of subsequent separation techniques. 17) Have you removed molecules which can be problematic for some screening approaches? A number of common components of plant extracts, including saponins, tannins and fatty acids, have been reported to cause interference in some biological assays. There is an argument for the pre-treatment of samples to remove such agents before library assembly. However, this also results in the loss of many compounds with potentially useful activity. To balance potential interference with compound diversity, the library has been pre-fractionated into one polar and one non-polar pre-fraction per extract without pre-treatment to remove major classes of phytochemicals.

 

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